Use of an 'in situ' disulfide cross-linking strategy to study ligand-dependent conformational changes in a class I GPCR[unreadable] Class I GPCRs represent by far the largest GPCR subfamily containing 670 full-length human receptor proteins. Members of this receptor family are the target of an extraordinarily large number of clinically important drugs. To study the molecular mechanisms underlying the activation of this class of receptors, we have used the M3 muscarinic acetylcholine receptor (M3 mAChR), a prototypic class I GPCR, as a model system.[unreadable] To monitor ligand-induced changes in GPCR structure, we employed an in situ disulfide cross-linking strategy that allows the detection of disulfide bond formation between Cys residues that are adjacent to each other in the three-dimensional (3D) structure of the receptor. One major advantage of this strategy is that ligand-dependent conformational changes can be detected in receptors present in their native membrane environment, without the need for any receptor purification and reconstitution steps. Specifically, we first generated a modified version of the M3 mAChR that lacked most endogenous Cys residues and contained a factor Xa cleavage site within the third intracellular loop (M3-Xa receptor). This modified receptor exhibits ligand binding and G protein coupling properties similar to those of the wild-type M3 mAChR. We next introduced pairs of Cys residues into the M3-Xa receptor background and used an disulfide cross-linking approach to examine whether the introduced Cys residues were able to form a disulfide bond with each other, either in the absence or presence of muscarinic ligands. [unreadable] During the past several years, we used this disulfide cross-linking strategy to analyze more than 100 different double Cys mutant M3-Xa receptors transiently expressed in COS-7 cells. These studies have led to novel insights into the conformational changes associated with M3 mAChR activation. The observed structural changes are predicted to involve multiple receptor regions, primarily distinct segments of transmembrane (TM) helices III, VI, and VII, as well as helix 8 (Wess et al. Trends Pharmacol. Sci., in press).[unreadable] During the past year, we examined whether different classes of muscarinic ligands (full versus inverse muscarinic agonists) had different effects on the relative orientation of helix 8 relative to the C-terminus of TM I. Helix 8 represents a cytoplasmic alpha-helical extension of TM VII to which it is connected via a short linker sequence. Considerable evidence suggests that helix 8 plays an important role in productive receptor/G protein coupling. The high-resolution structure of bovine rhodopsin indicates that several residues contained within helix 8 are located close to the cytoplasmic end of TM I. We therefore hypothesized that Cys residues substituted into this segment of TM I might serve as useful reporters to detect potential ligand-induced movements of helix 8 in disulfide cross-linking studies. We generated twenty double Cys mutant M3-Xa receptors, all of which contained one Cys substitution within the cytoplasmic end of TM I (A91-N95) and a second one within the N-terminal segment of helix 8 (K548-R551). We demonstrated that muscarinic agonists inhibited disulfide cross-linking in the A91C/T549C and F92C/F550C double Cys mutant M3 receptors (Li et al. J. Biol. Chem. 282, 26284-93, 2007). In contrast, atropine and NMS, two inverse muscarinic agonists, enhanced disulfide bond formation in these two mutant receptors (Li et al. J. Biol. Chem. 282, 26284-93, 2007). Our data therefore strongly support a model in which full muscarinic agonists trigger a separation of the N-terminal segment of helix 8 from the cytoplasmic end of TM I, thus preventing the formation of disulfide cross-links between Cys residues introduced at positions 91/549 and 92/550. On the other hand, inverse muscarinic agonists are predicted to decrease the distance between the cytoplasmic end of TM I and the N-terminal portion of helix 8.[unreadable] In a related cross-linking study (Li et al. Biochemistry 47, 2776-88, 2008), we examined whether the cytoplasmic end of TM V, a region known to be critically involved in receptor/G protein coupling, undergoes a major conformational change during receptor activation. Another goal was to determine and compare the disulfide cross-linking patterns observed after treatment of the different mutant receptors with full versus inverse muscarinic agonists. Specifically, we generated twenty double Cys mutant M3-Xa receptors harboring one Cys substitution within the cytoplasmic end of TM V (L249-I253) and a second one within the cytoplasmic end of TM VI (A489-L492) (Li et al. Biochemistry 47, 2776-88, 2008). Our cross-linking data strongly suggested that M3 receptor activation does not trigger major structural disturbances within the cytoplasmic segment of TM V, in contrast to the pronounced structural changes predicted to occur at the cytoplasmic end of TM VI. We also demonstrated that full and inverse muscarinic agonists had distinct effects on the efficiency of disulfide bond formation in specific double Cys mutant M3 receptors (Li et al. Biochemistry 47, 2776-88, 2008).[unreadable] The findings described above provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. Since all class I GPCRs are predicted to share a similar transmembrane topology, the conclusions drawn from this work should be of broad general relevance.